2014-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/687158摘要：細胞凋亡機制可能在濾泡成長、成熟及排卵的過程扮演重要的角色。我們初步的研究結果顯示BCL10(一個具有調控細胞凋亡能力的蛋白質具有caspase recruitment domain)會表現於老鼠的卵巢組織中的granulosa cells，我們也發現在人類granulosa cells 抑制BCL10 的表現會造成細胞週期停滯的現象。在本三年期計畫中我們主要將探討BCL10在濾泡成長、成熟及排卵等過程中的調控及其扮演的角色。本計畫的分年目標與執行策略分別為:第一年， 以動物模式探討自然月經週期情況下BCL10 在卵巢組織表現的情況，主要使用的方法為免疫組織染色，我們也將利用人類granulosa cells 探討生殖內分泌相關荷爾蒙對BCL10 產生的效應，並運用化學抑制劑探討個別的調控分子機制。第二年， 主要將探討BCL10 調控的訊息傳遞對濾泡成長的影響，主要的研究策略與方法是利用RNA 干擾技術抑制BCL10 在granulosa cells 的表現，配合細胞凋亡的細胞分子生物學方法探討BCL10 對NF-kB 活化的影響，並運用kinaseactivation array 及 transcription factor array 配合化學抑制劑及decoy ODN 策略探討BCL10 dependent kinase activation。第三年， 主要將探討BCL10 在活體內對濾泡成長、成熟及排卵等過程的效應，我們已經著手在建立卵巢組織專一性BCL10 抑制基因轉殖鼠，主要運用ovarian-specific promoter. 1 (OSP-1) 調控卵巢組織專一性，並以tetracyclin 調控bcl10 shRNA 表現，我們將以此基因轉殖鼠模式探討BCL10 在活體內對濾泡成長、成熟及排卵等過程的效應。我們也將嘗試以基因治療方式探討BCL10表現對濾泡成長、成熟及排卵等過程的效應。本研究成果將能證明具有調控細胞凋亡能力的 BCL10 對濾泡成長、成熟及排卵等過的重要性，也提供生殖相關荷爾蒙對BCL10 的調控機制，並釐清BCL10 基因治療對排卵機制的影響。<br> Abstract: Cell apoptosis during the follicular growth or maturation may play certain role inovulation. Our preliminary data revealed that BCL10 which is an apoptotic regulatory protein,containing a caspase recruitment domain is expressed in mouse ovary tissue, furthermoreinhibition of bcl10 gene expression caused a cell cycle arrest phenomenon. In this project, wewill investigate the role and the regulation of BCL10 in follicle maturation and ovulation.The research designs of the three years project are as follows. In the first year, the aim isto evaluate the expression of BCL10 in ovary in natural cycle in a mouse model byimmunohistochemical staining and to clarify the effect of pregnancy related endocrinemolecular estradiol, and progesterone on the regulation of BCL10 in human granulosa cells,furthermore, molecular biology methodologies will be used to clarify the signalingtransduction pathways and transcriptional regulation. In the second year, the aim is to clarifythe anti-apoptosis role and the effect of BCL10 on ovulation, BCL10 containing a caspaserecruitment domain, the anti-apoptosis role of BCL10 will be verified by series apoptosisassays, the effect of BCL10 on NF-kB activation will be verified by florescence stainingbased flow cytometry analysis. Other BCL10 dependent kinase activation and transcriptionfactor activation will be verified by array assays. In the third year, the aim is to verify the roleof BCL10 on follicle maturation in vivo. We will establish an ovary specific and BCL10conditional know-down mouse strain by using ovarian-specific promoter. 1 (OSP-1) [10]-Tetracycline-controlled BCL10 shRNA transcriptional activation strategy. The transgenicmouse will be used to evaluate the effect of BCL10 on follicle maturation in vivo byhistopathology judgment. Adenovirus-mediated BCL10 gene-therapy will be used to test therecover effect in the BCL10 knock-down mice and evaluate the function by histopathologyjudgment. Results obtained from this project may provide important information for theclinically relevant role in enhancing ovulation.